The present invention relates to two novel genes coding for xcex2-agarases and to their use for producing agar biodegradation enzymes.
It further relates to the Cytophaga drobachiensis strain from which these genes were isolated.
The sulfated galactans of Rhodophyceae, such as agars and carrageenans, represent the major polysaccharides of Rhodophyceae and are very widely used as gelling agents or thickeners in various branches of activity, especially the agri-foodstuffs sector. Approximately 6000 tonnes of agars and 22,000 tonnes of carrageenans are extracted annually from marine red algae for this purpose. Agars are produced industrially from red algae of the genera Gelidium and Gracilaria. Carrageenans are widely extracted from the genera Chondrus, Gigartina and Euchema.
Agaro-colloids are polysaccharide complexes consisting mainly of agars and agaroids. Each agaro-colloid has a different content of each of the above compounds, so its gelling strength is different. Agar gel comprises a matrix of double-helix polymer chains held together by hydrogen bonds.
There are two types of enzyme capable of degrading agars: xcex1-agarases and xcex2-agarases. xcex2-Agarases act on the xcex2-1,4 linkage and xcex1-agarases act on the xcex1-1,3 linkage.
Microorganisms which produce enzymes capable of hydrolyzing agars have already been isolated. This capacity to digest agar has been attributed to the genera Pseudomonas (MORRICE et al., Eur. J. Biochem. 137, 149-154, (1983)), Streptomyces (HODGSON and CHATER, J. Gen. Microbiol. 124, 339-348, (1981)), Cytophaga (VAN DER MEULEN and HARDER, J. Microbiol. 41, 431-447, (1975)) and Vibrio (SUGANO et al., Appl. Environ. Microbiol. 59, 1549-1554, (1993)). Several xcex2-agarase genes have already been isolated. Thus BELAS et al. isolated the gene of an agarase from Pseudomonas atlantica (Appl. Environ. Microbiol. 54, 30-37, (1988)). BUTTNER et al. isolated an agarase from Streptomyces coelicolor and sequenced the corresponding gene (Mol. Gen. Genet. 209, 101-109, (1987)). SUGANO et al. cloned and sequenced two different agarase genes from Vibrio sp. JT0107, which they called agaA (Appl. Environ. Microbiol. 59, 3750-3756, (1993)) and agaB (Biochimica et Biophysica Acta 1 218, 105-108, (1994)).
The Applicant has now isolated, from the red alga Delesseria sanguinea, a bacterial strain which has agarase activity.
This strain was deposited in the DSMZ Collection (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures)) on May 8, 1998 under the number DSM 12170. It forms the first subject of the present invention.
Taxonomic investigation of this strain, performed by techniques well known to those skilled in the art, shows that it belongs to the genus Cytophaga (bacteria of the CFB or xe2x80x9cCytophaga/Flexibacter/Bacteroidesxe2x80x9d group). In fact, this strain develops by spreading and has yellow colonies encrusted in the agar, which is then liquefied. The bacterium is a Gram-negative bacterium and has the shape of a non-mobile rod of 0.3-0.4xc3x973.0-8.0 xcexcmxc3x97xcexcm. When a drop of culture of the strain is inoculated at the center of an agar dish, the colony develops with concentric growth of the margin and this mobility is not inhibited by diethyl ether, which is an inhibitor of the flagellar apparatus. The strain is aerobic and has an oxidative metabolism. It produces flexirubin, which is a pigment rarely found in isolates of marine Cytophaga but present in non-marine Cytophaga. It is capable of assimilating various carbon sources and degrading several types of macromolecule such as agar, carrageenan, starch and gelatin.
The Applicant carried out an in-depth study to find out what species this strain belonged to. Thus it determined the percentage guanine and cytosine composition of the DNA of the strain of the invention and found that the values were between 43 and 49%. It also sequenced its 16S DNA by the method well known to those skilled in the art for finding out the taxonomic position of a strain (FOX et al., Int. J. Syst. Bacteriol. 22, 44-57, (1977)). The sequencing result shows that the strain of the invention is very similar to Cytophaga uliginosa. (There is a 99% similarity of sequence between the 16S DNA of C. uliginosa and that of the strain of the invention.) However, DNA/DNA hybridization between the two strains (45%) shows that they are different species.
Furthermore, the strain of the invention has similar morphological, biochemical and physiological characteristics to the strain Pseudomonas drobachiensis nov. comb. isolated by HUMM (Duke Univ. Mar. Stn. Bull. 3, 43-75, (1946)). It was therefore named Cytophaga drobachiensis. 
The Applicant also isolated two genes with xcex2-agarase activity from Cytophaga drobachiensis DSM 12170.
Thus the present invention further relates to the novel agaA gene coding for a xcex2-agarase, which has the DNA sequence SEQ ID No. 1.
It further relates to the novel agaB gene coding for a xcex2-agarase, which has the DNA sequence SEQ ID No. 3.
These two genes code for two different xcex2-agarases produced by C. drobachiensis DSM 12170, namely the xcex2-agarases called proteins AgaA and AgaB.
The present invention further relates to the nucleic acid sequences, namely the genomic DNA sequences and the DNA or mRNA sequences, which comprise or consist of a concatenation of nucleotides coding for the protein AgaA or the protein AgaB or for any one of their peptide fragments as defined below.
The invention therefore relates to:
All the nucleic acid sequences coding for the protein AgaA in its entirety or for one or more of its peptide fragments. These sequences are preferably represented by:
a) the DNA sequence SEQ ID No. 1 coding for the protein AgaA, and its fragments coding for the peptide fragments of said protein;
b) the DNA sequences which hybridize under specific stringency conditions with the above sequence or one of its fragments;
c) the DNA sequences which, because of the degeneracy of the genetic code, are derived from one of the sequences a) and b) above and code for the protein AgaA or the fragments of said protein; and
d) the corresponding mRNA sequences.
All the nucleic acid sequences coding for the protein AgaB in its entirety or for one or more of its peptide fragments. These sequences are preferably represented by:
a) the DNA sequence SEQ ID No. 3 coding for the protein AgaB, and its fragments coding for the peptide fragments of said protein;
b) the DNA sequences which hybridize under specific stringency conditions with the above sequence or one of its fragments;
c) the DNA sequences which, because of the degeneracy of the genetic code, are derived from one of the sequences a) and b) above and code for the protein AgaB or the fragments of said protein; and
d) the corresponding mRNA sequences.
The present invention further relates to the nucleic acid sequence SEQ ID No. 5 coding for the specific peptide fragment AgaAxe2x80x2, which will be described below. This sequence corresponds to nucleic acids 223-1050 of SEQ ID No. 1.
The invention therefore further relates to the nucleic acid sequences coding for said peptide fragment AgaAxe2x80x2 and its peptide fragments, which are represented by:
a) the DNA sequence SEQ ID No. 5 coding for the peptide fragment AgaAxe2x80x2, and its fragments coding for the peptide fragments of said peptide fragment AgaAxe2x80x2;
b) the DNA sequences which hybridize under specific stringency conditions with the above sequence or one of its fragments;
c) the DNA sequences which, because of the degeneracy of the genetic code, are derived from one of the sequences a) and b) above and code for the peptide fragment AgaAxe2x80x2 or the fragments of said fragment; and
d) the corresponding mRNA sequences.
The nucleic acids according to the invention can be prepared by chemical synthesis or genetic engineering using the techniques well known to those skilled in the art and described for example in SAMBROOK et al. (xe2x80x9cMolecular Cloning: a Laboratory Manualxe2x80x9d, published by Cold Spring Harbor Press, N.Y., 1989).
For example, the DNA sequences according to the invention can be synthesized by amplifying the genes of Cytophaga drobachiensis by the PCR (polymerase chain reaction) method, as described for example by GOBLET et al. (Nucleic Acid Research 17, 2144, (1989)), using, as primers, synthetic oligonucleotides defined from the DNA sequence SEQ ID No. 1 or SEQ ID No. 3.
The nucleic acid fragment amplified in this way can then be cloned into an expression vector by the techniques described in MANIATIS et al. (Molecular Cloning. A laboratory manual, New York (1982)).
The invention further relates to the prokaryotic cells and eukaryotic cells transformed with the aid of an expression vector containing a nucleic acid sequence according to the invention. This expression vector, which can be e.g. in the form of a plasmid, must contain, in addition to the nucleic acid sequence of the invention, the means necessary for its expression, such as, in particular, a promoter, a transcription terminator, an origin of replication and, preferably, a selection marker. The transformation of prokaryotic cells and eukaryotic cells is a technique well known to those skilled in the art, who will easily be able to determine, as a function of the microorganism to be transformed, the means necessary for expression of the DNA sequence according to the invention.
The preferred prokaryotic microorganisms for the purposes of the invention are Escherichia coli and Bacillus subtilis. 
The cells of Aspergillus niger, Trichoderma viridae or Pichia pastoris may be mentioned in particular as examples of eukaryotic cells which are suitable for the purposes of the invention.
The present invention further relates to the novel protein AgaA of C. drobachiensis, which comprises SEQ ID No. 2.
It further relates to the novel protein AgaB of C. drobachiensis, which comprises SEQ ID No. 4.
The novel protein AgaA is composed of 539 amino acids and has a theoretical molecular weight of 60.001 kDa. After removal of the signal peptide, this protein has a calculated molecular weight of 57.768 kDa.
The novel protein AgaB is composed of 353 amino acids. After removal of the signal peptide, this protein has a calculated molecular weight of 40.680 kDa.
The present invention further relates to the peptide fragments of the proteins AgaA and AgaB which result from the addition, suppression and/or replacement of one or more amino acids, said peptide fragments having conserved the xcex2-agarase activity.
The present invention further relates to the peptide fragment AgaAxe2x80x2, which has SEQ ID No. 6.
This peptide fragment AgaAxe2x80x2, composed of 276 amino acids, corresponds to amino acids 20-295 of the protein AgaA.
The invention further relates to the peptide fragments of AgaAxe2x80x2 which result from the addition, suppression and/or replacement of one or more amino acids, said peptide fragments having conserved the xcex2-agarase activity.
The proteins and peptide fragments according to the invention can be obtained by the techniques of genetic engineering comprising the following steps:
culture of prokaryotic cells or eukaryotic cells transformed by an expression vector possessing a nucleic acid sequence according to the invention; and
recovery of the protein or peptide fragment produced by said cells.
These techniques are well known to those skilled in the art and further details may be obtained by reference to the following work: Recombinant DNA Technology I, editors: Ales Prokop, Raskesh K. Bajpai; Annals of the New York Academy of Sciences, volume 646, 1991.
The peptide fragments can also be prepared by conventional chemical peptide synthesis well known to those skilled in the art.
The invention will now be described in detail with the aid of the experimental section below.
The techniques described in these Examples, which are well known to those skilled in the art, are largely explained in detail in the work by SAMBROOK et al. (supra) or in the work by MANIATIS et al. (supra).